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目的:在转基因小鼠中,研究丙肝病毒(HCV)核心蛋白激活诱导型一氧化氮合成酶(iNOS)表达对DNA的损伤作用。方法:携带HCV cDNA的质粒载体pSG-1/HCV显微注射入C57小鼠受精卵。取12月龄小鼠肝组织标本,RT-PCR和W estern印迹检测HCV和iNOS的表达水平。尾静脉注射小分子RNA干扰片段作为实验组,PBS作为对照组,对比各组小鼠的iNOS在mRNA和蛋白水平的表达情况及其体内NO的含量。通过连接反应介导的PCR(LM-PCR)方法,评价iNOS和NO水平变化对DNA双链断裂(DSB)的影响。结果:在转基因小鼠的肝脏中,有HCV和iNOS的特异性高表达,NO含量和DSB水平也远高于未转染HCV基因的正常小鼠。经RNA干扰作用6 d后,实验组小鼠的iNOS表达在mRNA和蛋白水平都有非常明显的下降(P<0.01);同时NO含量也下降(P<0.01),DSB现象基本消失。结论:HCV核心蛋白在肝细胞中能激活iNOS的表达,诱导生成NO,进而引发DSB现象,造成对基因组DNA的损伤。RNA干扰可以有效逆转HCV对iNOS基因的激活,对于治疗和预防HCV患者发生的肝癌具有重要意义。
OBJECTIVE: To study the DNA damaging effect of hepatitis C virus (HCV) core protein activation-inducible nitric oxide synthase (iNOS) in transgenic mice. Methods: Plasmid pSG-1 / HCV carrying HCV cDNA was microinjected into fertilized eggs of C57 mice. The liver tissues of 12-month-old mice were used to detect the expression of HCV and iNOS by RT-PCR and western blotting. The tail vein was injected with small RNA interference fragment as experimental group and PBS as control group. The expression of iNOS at mRNA and protein level and NO content in each group were compared. The effect of changes in iNOS and NO levels on DNA double-strand break (DSB) was evaluated by ligation-mediated PCR (LM-PCR). Results: HCV and iNOS were highly expressed in liver of transgenic mice, and the content of NO and the level of DSB were much higher than those in normal mice without HCV gene transfection. After 6 days of RNA interference, the expression of iNOS in the experimental group was significantly decreased at the mRNA and protein levels (P <0.01), and the content of NO was also decreased (P <0.01). The DSB phenomenon disappeared. CONCLUSION: HCV core protein can activate iNOS expression in hepatocytes, induce NO generation, and then lead to DSB phenomenon, resulting in damage to genomic DNA. RNA interference can effectively reverse HCV activation of iNOS gene, for the treatment and prevention of liver cancer patients with HCV is of great significance.